Strong adaptive immunity depends on a strong innate immune response triggered by pathogen-derived molecules, such as Toll-Like Receptor (TLR) ligands. Current cancer vaccines typically induce tumor-specific T cells but no tumor regression. This may be because tumors are deficient in TLR ligands, reducing innate immune activation in tumors and thereby limiting the priming, accumulation, in situ activation and anti-tumor effector function of tumor-specific T cells. Plasmacytoid Dendritic Cells (pDCs) are cells of the innate immune system that critically orchestrate both innate and adaptive immune responses. pDCs have been found inside human tumors and are therefore potentially ideal targets for therapeutic intervention with TLR7 and TLR9 agonists which activate pDCs. However, it is unknown whether intratumoral pDCs are activated or whether they can respond to TLR agonist stimulation in vivo, and what the downstream effects of such activation are for innate and adaptive anti-tumor immunity. In addition, little is known about the mechanism through which pDCs could stimulate innate and adaptive anti-tumor immunity in vivo. Since immune responses are complex, they can only be fully understood through in vivo studies. We have developed a mouse model that allows us to study the presence and function of pDCs in tumors in vivo. We found that non-activated pDCs were present in murine melanoma. TLR agonist-activated pDCs could activate conventional mDCs, enhancing their capacity to prime tumor-specific T cells in vivo. Furthermore, activated pDCs potently activated NK cells in vivo to induce tumor destruction and de novo priming of additional tumorspecific CD8+ T cells. Building on the gplOO-specific TCR transgenic pmel-1 mouse model we have previously developed, we also found that in vivo activation of pDCs with TLR agonists enhanced tumor regression mediated by vaccine-induced, gplOO-specific CD8+ T cells. Based on these data we hypothesize that activation of intratumoral pDCs by TLR agonists in vivo will result in activation of mDCs and NK cells in the tumor leading to the induction of potent innate and adaptive anti-tumor immune responses. To test this hypothesis we will activate intratumoral pDCs in vivo with TLR agonists and test their ability to activate mDCs and NK cells and to induce the accumulation of tumor-specific T cells and tumor destruction, through the following Specific Aims: Specific Aim 1. Characterize the ability of intratumoral pDCs to interact with mDCs. Specific Aim 2. Evaluate the interactions between intratumoral pDCs and NK cells. Specific Aim 3. Determine whether intratumoral pDC activation by TLR agonists enhances T cellmediated anti-tumor function. Our goal is to identify principles which may be generalized towards improving immunotherapy of cancer patients.